br After addition of media containing the compounds to
After addition of media containing the compounds to be tested, plates were incubated for 24 h under standard conditions as described previously [25,27]. NHDF and Me45 ITF2357 (Givinostat) were washed three times with saline (1xPBS; PAA), 50 μl of MTT solution (3-[4,5-di-methylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma) in phenol red free DMEM-F12 (PAA) were added into each well, and the plates were incubated for 2 h until the colour in the control wells had changed from yellow to purple, than the formazan crystals were dissolved in isopropanol:HCl solution (v/v 1:1). The absorbance was measured at a wavelength of λ = 570 nm using a microplate spectrophotometer
J. Nackiewicz et al.
MTS antiproliferative assay were performed after producer protocol (Promega), where 20 μl of MTS dye [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] was added to the PBS washed Me45 cells and incubated for 3 h in darkness. The optical product were qualitatively measured, with the absorbance at 490 nm (Epoch; BioTek, Winooski, VT, USA).
2.3.4. Flow Cytometry Analyses for Apoptosis and ROS Production Me45 cells were washed and collected by trypsinization after 15 min and 24 h of incubation after compounds addition and light irradiation, centrifuged (1500 rpm, 3 min), and stained according to the Annexin-V apoptosis assay  or the ROS assay procedures [25,27]. For apop-tosis assays cell pellets were resuspended in 50 μl of cold Annexin-V labelling buffer, and then 2.5 μl of FITC-labeled Annexin-V antibody was added (BioLegend, San Diego, Cal., USA) followed by 10 μl of propidium iodide (PI) solution (100 μg/ml; Sigma). After 20 min of incubation in darkness, 250 μl of Annexin-V labelling buffer was added and the samples were incubated on ice and in the dark for 15 min. Flow cytometric analyses were performed immediately using an Aria III flow cytometer (Becton Dickinson) with the FITC configuration (488 nm excitation; emission: LP mirror 503, BP filter 530/30) or PE config-uration (547 nm excitation; emission: 585 nm) and at least 10,000 cells were counted.
ROS levels were measured by flow cytometry using specific dye. For global ROS level 2′,7′-dichlorofluorescin diacetate (DCFH-DA; Sigma) was used, a cell-permeable non-fluorescent probe which converts into the highly fluorescent 2′,7′-dichlorofluorescein (DCF) upon deacetyla-tion by intracellular esterases and oxidation by ROS. Three hundred μl of cell suspension in 1xPBS (prepared in triplicates) were stained with DCFH-DA (30 μM). After incubation in darkness (30 min at 37 °C) the cells were washed with PBS and kept for
15 min on ice. Then, the fluorescence was measured using a flow cytometer with the FITC configuration (488 nm excitation; emission: LP mirror 503, BP filter 530/30) and at least 10,000 cells were counted. The results were analyzed using the free software FlowingSoftware 2.5.0 (Perttu Terho, Turku Centre for Biotechnology, University of Turku, Finland) and are presented as mean fluorescence.
The samples prepared for flow cytometry measurements were also used for live-cell imaging. Representative samples were transferred into 4-chamber microscope slides (Becton Dickinson) and the medium was replaced by fresh medium containing ZnPcOC (30 μM) for 72 h. Live observation using the inverted Cell R epi-fluorescence system (Olympus) allowed imaging of intracellular ZnPcOC accumulation. Images were taken from transit channels at magnification 200 x, using ImageJ 1.47v (Wayne Rasband, National institute of Health, USA).
2.4. Statistical Analysis
The results are expressed as means ± SD from three separate ex-periments, performed in quadruplicates (n = 12) for cytotoxicity and triplicates (n = 3) for cytometric assays. Statistical significance vs the control was calculated by t-tests and p < .05 is indicated by star (*). The results were analyzed using MS Office ver. 2.5.0 and MS Excel 2007.